[Yeasts from the CTG clade (Candida clade): Biology, impact in human health, and biotechnological applications]. / Les levures du clade CTG (clade Candida) : biologie, incidence en santé humaine et applications en biotechnologie.

Among the subdivision of Saccharomycotina (ascomycetes budding yeasts), the CTG clade (formerly the Candida clade) includes species that display a particular genetic code. In these yeasts, the CTG codon is predominantly translated as a serine instead of a leucine residue. It is now well-known that some CTG clade species have a major impact on human and its activities. Some of them are recognized as opportunistic agents of fungal infections termed candidiasis. In addition, another series of species belonging to the CTG clade draws the attention of some research groups because they exhibit a strong potential in various areas of biotechnology such as biological control, bioremediation, but also in the production of valuable biocompounds (biofuel, vitamins, sweeteners, industrial enzymes). Here we provide an overview of recent advances concerning the biology, clinical relevance, and currently tested biotechnological applications of species of the CTG clade. Future directions for scientific research on these particular yeasts are also discussed.

Prequels to Synthetic Biology: From Candidate Gene Identification and Validation to Enzyme Subcellular Localization in Plant and Yeast Cells.

Natural compounds extracted from microorganisms or plants constitute an inexhaustible source of valuable molecules whose supply can be potentially challenged by limitations in biological sourcing. The recent progress in synthetic biology combined to the increasing access to extensive transcriptomics and genomics data now provide new alternatives to produce these molecules by transferring their whole biosynthetic pathway in heterologous production platforms such as yeasts or bacteria. While the generation of high titer producing strains remains per se an arduous field of investigation, elucidation of the biosynthetic pathways as well as characterization of their complex subcellular organization are essential prequels to the efficient development of such bioengineering approaches. Using examples from plants and yeasts as a framework, we describe potent methods to rationalize the study of partially characterized pathways, including the basics of computational applications to identify candidate genes in transcriptomics data and the validation of their function by an improved procedure of virus-induced gene silencing mediated by direct DNA transfer to get around possible resistance to Agrobacterium-delivery of viral vectors. To identify potential alterations of biosynthetic fluxes resulting from enzyme mislocalizations in reconstituted pathways, we also detail protocols aiming at characterizing subcellular localizations of protein in plant cells by expression of fluorescent protein fusions through biolistic-mediated transient transformation, and localization of transferred enzymes in yeast using similar fluorescence procedures. Albeit initially developed for the Madagascar periwinkle, these methods may be applied to other plant species or organisms in order to establish synthetic biology platform.

Virus-induced gene silencing in Catharanthus roseus by biolistic inoculation of tobacco rattle virus vectors.

Catharanthus roseus constitutes the unique source of several valuable monoterpenoid indole alkaloids, including the antineoplastics vinblastine and vincristine. These alkaloids result from a complex biosynthetic pathway encompassing between 30 and 50 enzymatic steps whose characterisation is still underway. The most recent identifications of genes from this pathway relied on a tobacco rattle virus-based virus-induced gene silencing (VIGS) approach, involving an Agrobacterium-mediated inoculation of plasmids encoding the two genomic components of the virus. As an alternative, we developed a biolistic-mediated approach of inoculation of virus-encoding plasmids that can be easily performed by a simple bombardment of young C. roseus plants. After optimisation of the transformation conditions, we showed that this approach efficiently silenced the phytoene desaturase gene, leading to strong and reproducible photobleaching of leaves. This biolistic transformation was also used to silence a previously characterised gene from the alkaloid biosynthetic pathway, encoding iridoid oxidase. Plant bombardment caused down-regulation of the targeted gene (70%), accompanied by a correlated decreased in MIA biosynthesis (45-90%), similar to results obtained via agro-transformation. Thus, the biolistic-based VIGS approach developed for C. roseus appears suitable for gene function elucidation and can readily be used instead of the Agrobacterium-based approach, e.g. when difficulties arise with agro-inoculations or when Agrobacterium-free procedures are required to avoid plant defence responses.

Cloning and expression of cDNAs encoding two enzymes of the MEP pathway in Catharanthus roseus.

Two periwinkle cDNAs (crdxr and crmecs) encoding enzymes of the non-mevalonate terpenoid pathway were characterized using reverse transcription-PCR strategy based on the design of degenerated oligonucleotides. The deduced amino acid sequence of crdxr is homologue to 1-deoxy-D-xylulose 5-phosphate reductoisomerases. Crmecs represents the first plant cDNA encoding a protein similar to the 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase from Escherichia coli. Expression of crdxr and crmecs genes was up-regulated in periwinkle cells producing monoterpenoid indole alkaloids. Involvement of the 2C-methyl-D-erythritol 4-phosphate pathway in alkaloid biosynthesis is discussed.

Purification and Characterization of Cinnamoyl-Coenzyme A:NADP Oxidoreductase in Eucalyptus gunnii.

Cinnamoyl-coenzyme A:NADP oxidoreductase (CCR, EC 1.2.1.44), the entry-point enzyme into the monolignol biosynthetic pathway, was purified to apparent electrophoretic homogeneity from differentiating xylem of Eucalyptus gunnii Hook. The purified protein is a monomer of 38 kD and has an isoelectric point of 7. Although Eucalyptus gunnii CCR has approximately equal affinities for all possible substrates (p-coumaroyl-coenzyme A, feruloyl-coenzyme A, and sinapoyl-coenzyme A), it is approximately three times more effective at converting feruloyl-coenzyme A than the other substrates. To gain a better understanding of the catalytic regulation of Eucalyptus CCR, a variety of compounds were tested to determine their effect on CCR activity. CCR activity is inhibited by NADP and coenzyme A. Effectors that bind lysine and cysteine residues also inhibit CCR activity. As a prerequisite to the study of the regulation of CCR at the molecular level, polyclonal antibodies were obtained.

Purification and Characterization of Geranyl Diphosphate Synthase from Vitis vinifera L. cv Muscat de Frontignan Cell Cultures.

A geranyl diphosphate synthase (EC 2.5.1.1), which catalyzes the formation of geranyl diphosphate from dimethylallyl diphosphate and isopentenyl diphosphate, was isolated from Vitis vinifera L. cv Muscat de Frontignan cell cultures. Purification of the enzyme was achieved successively by ammonium sulfate precipitation and chromatography on DEAE-Sephacel, hydroxylapatite, Mono Q, Phenyl Superose, Superose 12, and preparative nondenaturing polyacrylamide gels. The enzyme formed only geranyl diphosphate as a product. In all cases, neither neryl diphosphate, the cis isomer, nor farnesyl diphosphate was detected. The enzyme showed a native molecular mass of 68 [plus or minus] 5 kD as determined by gel permeation. On sodium dodecyl sulfate polyacrylamide gels, geranyl diphosphate synthase purified to electrophoretic homogeneity migrated with a molecular mass of 66 [plus or minus] 2 kD. Michaelis constants for isopentenyl diphosphate and dimethylallyl diphosphate were 8.5 and 56.8 [mu]M, respectively. The enzyme required Mn2+ and Mg2+ as cofactors and its activity was enhanced by Triton X-100. Inorganic pyrophosphate, aminophenylethyl diphosphate, and geranyl diphosphate had inhibitory effects on the enzyme.

Evidence for a geranyl-diphosphate synthase located within the plastids of Vitis vinifera L. cultivated in vitro.

Intact plastids from cell suspensions of Vitis vinifera L. cv. Muscat de Frontignan, free of detectable contamination by other particles as judged by the distribution of organelle-specific marker enzymes and by electron microscopy, exhibit geranyl-diphosphate synthase activity (EC 2.5.1.1). This synthase activity remains stable after tryptic digestion of unlysed organelles and is enhanced by plastid disruption. We conclude that the enzyme is located within the organelle. The possibility of an isopentenyl diphosphate/dimethylallyl diphosphate translocating system which would play a major role in the regulation of monoterpene metabolism is discussed.

Prenyltransferase compartmentation in cells of Vitis vinifera cultivated in vitro.

Two prenyltransferases were located in cell cultures of Vitis vinifera. A geranyl pyrophosphate synthase (EC 2.5.1.1) was associated with plastid-like membranes whereas a farnesyl pyrophosphate synthase (EC 2.5.1.10) was found to be soluble.